Fat malabsorption in cystic fibrosis patients receiving enzyme replacement therapy is due to impaired intestinal uptake of long-chain fatty acids

BACKGROUND: Recombinant human growth hormone (GH) improves in vivo cardiac function in rats with postinfarction heart failure (MI). We examined the effects of growth hormone (14 days of 3.5 mg. kg-1. d-1 begun 4 weeks after MI) on contractile reserve in left ventricular myocytes from rats with chronic postinfarction heart failure. METHODS AND RESULTS: Cell shortening and [Ca2+]i were measured with the indicator fluo 3 in myocytes from MI, MI+GH, control, and normal animals treated with GH (C+GH) under stimulation at 0.5 Hz at 37 degrees C. Cell length was similar in MI and MI+GH rats (150+/-5 and 157+/-5 microm) and was greater in these groups than in the control and C+GH groups (140+/-4 and 139+/-4 microm, P<0.05). At baseline perfusate calcium of 1.2 mmol/L, myocyte fractional shortening and [Ca2+]i transients were similar among the 4 groups. We then assessed contractile reserve by measuring the increase in myocyte fractional shortening in the presence of high-perfusate calcium of 3.5 mmol/L. In the control and C+GH groups, myocyte fractional shortening and peak systolic [Ca2+]i were similarly increased in the presence of high-perfusate calcium. In the presence of high-perfusate calcium, both myocyte fractional shortening and peak systolic [Ca2+]i were depressed in the MI compared with the control groups. In contrast, myocyte fractional shortening (14.1+/-.9% versus 11.1+/-.9%, P<0.05) and peak systolic [Ca2+]i (647+/-43 versus 509+/-37 nmol/L, P<0.05) were significantly higher in MI+GH than in MI rats and were comparable to controls. Left ventricular myocyte expression of sarcoplasmic reticulum Ca2+ ATPase 2 (SERCA-2) and left ventricular SERCA-2 protein levels were increased in MI+GH compared with MI rats. CONCLUSIONS: Calcium-dependent contractile reserve is depressed in myocytes from rats with postinfarction heart failure. Long-term growth hormone therapy increases contractile reserve by restoring normal augmentation of systolic [Ca2+]i in myocytes from rats with postinfarction heart failure.     

Propofol and ketamine only inhibit intracellular Ca2+ transients and contraction in rat ventricular myocytes at supraclinical concentrations

BACKGROUND: The cellular mechanisms that mediate the cardiodepressant effects of intravenous anesthetic agents remain undefined. The objective of this study was to elucidate the direct effects of propofol and ketamine on cardiac excitation-contraction coupling by simultaneously measuring intracellular calcium concentration ([Ca2+]i) and shortening in individual, field-stimulated ventricular myocytes. METHODS: Freshly isolated rat ventricular myocytes were loaded with the Ca2+ indicator, fura-2, and placed on the stage of an inverted fluorescence microscope in a temperature-regulated bath. [Ca2+]i and myocyte shortening (video edge detection) were monitored simultaneously in individual cells that were field-stimulated at 0.3 Hz. RESULTS: Baseline [Ca2+]i (mean +/- SEM) was 80 +/- 12 nM, and resting cell length was 112 +/- 2 microm. Field stimulation increased [Ca2+]i to 350 +/- 23 nM, and the myocytes shortened by 10% of diastolic cell length. Both intravenous anesthetic agents caused dose-dependent decreases in peak [Ca2+]i and shortening. At 300 microM, propofol prolonged time to peak concentration and time to 50% recovery for [Ca2+]i and shortening. In contrast, changes in time to peak concentration and time to 50% recovery in response to ketamine were observed only at the highest concentrations. Neither agent altered the amount of Ca2+ released from intracellular stores in response to caffeine. Propofol but not ketamine, however, caused a leftward shift in the dose-response curve to extracellular Ca2+ for shortening, with no concomitant effect on peak [Ca2+]i. CONCLUSIONS: These results indicate that both intravenous anesthetic agents have a direct negative inotropic effect, which is mediated by a decrease in the availability of [Ca2+]i. Propofol but not ketamine may also alter sarcoplasmic reticulum Ca2+ handling and increase myofilament Ca2+ sensitivity. The effects of propofol and ketamine are primarily apparent at supraclinical concentrations, however.     

Effects of the nitric oxide donor sodium nitroprusside on intracellular pH and contraction in hypertrophied myocytes

BACKGROUND: We compared the effects of the nitric oxide donor sodium nitroprusside (SNP) on intracellular pH (pHi), intracellular calcium concentration ([Ca2+]i) transients, and cell contraction in hypertrophied adult ventricular myocytes from aortic-banded rats and age-matched controls. METHODS AND RESULTS: pHi was measured in individual myocytes with SNARF-1, and [Ca2+]i transients were measured with indo 1 simultaneously with cell motion. Experiments were performed at 37 degrees C in myocytes paced at 0.5 Hz in HEPES-buffered solution (extracellular pH = 7.40). At baseline, calibrated pHi, diastolic and systolic [Ca2+]i values, and the amplitude of cell contraction were similar in hypertrophied and control myocytes. Exposure of the control myocytes to 10(-6) mol/L SNP caused a decrease in the amplitude of cell contraction (72 +/- 7% of baseline, P < .05) that was associated with a decrease in pHi (-0.10 +/- 0.03 U, P < .05) with no change in peak systolic [Ca2+]i. In contrast, in the hypertrophied myocytes exposure to SNP did not decrease the amplitude of cell contraction or cause intracellular acidification (-0.01 +/- 0.01 U, NS). The cGMP analogue 8-bromo-cGMP depressed cell shortening and pHi in the control myocytes but failed to modify cell contraction or pHi in the hypertrophied cells. To examine the effects of SNP on Na(+)-H+ exchange during recovery from intracellular acidosis, cells were exposed to a pulse and washout of NH4Cl. SNP significantly depressed the rate of recovery from intracellular acidosis in the control cells compared with the rate in hypertrophied cells. CONCLUSIONS: SNP and 8-bromo-cGMP cause a negative inotropic effect and depress the rate of recovery from intracellular acidification that is mediated by Na(+)-H+ exchange in normal adult rat myocytes. In contrast, SNP and 8-bromo-cGMP do not modify cell contraction or pHi in hypertrophied myocytes.     

Effects of angiotensin II on inotropy and intracellular Ca2+ handling in normal and hypertrophied rat myocardium

The direct inotropic effect of angiotensin II on the myocardium is still controversial and little information exists as to its potential modification by heart disorders. Therefore, this study performed simultaneous measurements of isometric force and intracellular Ca2+ concentrations ([Ca2+]i) in left ventricular papillary muscles from sham-operated and aortic-banded rats at 10 weeks post-surgery. Angiotensin II (10(-6) M) induced a reduction of peak systolic [Ca2+]i (0.56 +/- 0.03 to 0.48 +/- 0.04 microM; P<0.05) and a parallel but insignificant diminution of developed tension (10.5 +/- 1.3 to 9.6 +/- 0.8 mN/mm2) in normal papillary muscles from sham-operated animals. Hypertrophied papillary muscles from aortic-banded rats demonstrated a significant decline in both peak systolic [Ca2+]i (0.51 +/- 0.02 to 0.44 +/- 0.01 microM; P<0.05) and developed tension (8.4 +/- 1.1 to 6.8 +/- 1.7 mN/mm2; P<0.05) after addition of angiotensin II. The time courses of the mechanical contraction and the intracellular Ca2+ signal were prolonged by angiotension II in both groups. Isoproterenol dose-dependently increased developed tension and peak systolic [Ca2+]i in papillary muscles from sham-operated rats. In contrast, the positive inotropic response to isoproterenol was markedly reduced in hypertrophied muscles despite a seemingly unimpaired increase in peak systolic [Ca2+]i. Pretreatment with angiotensin II (10(-6) M) resulted in a significant attenuation of the systolic [Ca2+]i response to isoproterenol stimulation in both normal and hypertrophied papillary muscles. Neither the bradykinin B2 antagonist icatibent (10(-6) M) nor the nitric oxide (NO) inhibitor L-NMMA (10(-6) M) abolished the depressant effects of angiotension II. Thus, ANG II induces a parallel decline of the mechanical performance and Ca2+ availability in rat myocardium. These effects are more distinct in hypertrophied than in normal muscle and become accentuated during beta-adrenergic stimulation. The underlying mechanism is not associated with the NO pathway but might involve a negative functional coupling between the angiotensin and beta-adrenergic-receptor complex.     

Rapid effects of cytochalasin-D on contraction and intracellular calcium in single rat ventricular myocytes

Contraction and intracellular calcium ([Ca2+]i) transients were recorded using a video edge detector and fluorescence spectrophotometry, respectively, in rat ventricular myocytes at 22-24 degreesC stimulated at a frequency of 1 Hz. Application of the F-actin disrupter cytochalasin-D (Cyt-D) caused a large reduction in the amplitude of contraction and a small increase in the [Ca2+]i transient. These responses began within a few seconds of application and were complete after 2 min of exposure. Phase-plane relationships of contraction and [Ca2+]i were consistent with cytochalasin-D causing a decrease in myofilament responsiveness to Ca2+.     

Cytoplasmic sodium, calcium and free energy change of the Na+/Ca2+-exchanger in rat ventricular myocytes

The relationship between changing driving force of the Na+/Ca2+-exchanger (deltaG(exch)) and associated cytosolic calcium fluxes was studied in rat ventricular myocytes. DeltaG(exch) was abruptly reversed by the reduction of extracellular sodium ([Na+]o) with or without sustained depolarization by the elevation of potassium ([K+]o). Cytosolic sodium ([Na+]i) and calcium ([Ca2+]i) were measured with SBFI and indo-1 respectively and the time course of recovery of deltaG(exch) was calculated. Following abrupt reversal of deltaG(exch) from +4.1 to -9.2 kJ/mol [Na+]i exponentially decreased from 9.6-2.5 mmol/l (t(1/2) about 30 s) and [Ca2+]i transiently increased to a peak value after about 30 s. Negative values of deltaG(exch) were associated with an increase and positive values with a decrease of [Ca2+]i. Equilibrium (deltaG(exch) = 0) was reached after about 30 s coinciding with the time to peak [Ca2+]i. After 180 s deltaG(exch) reached a new steady state at +3.5 kJ/mol. Inhibition of SR with ryanodine or thapsigargin reduced the amplitude of the [Ca2+]i transient and shifted its peak to 80 s, but did not affect the time course of [Na+]i changes. In the presence of ryanodine or thapsigargin the time required for deltaG(exch) to recover to equilibrium was also shifted to 80 s. When we changed the deltaG(exch) to the same extent by the reduction of [Na+]o in combination with a sustained depolarization, [Na+]i decreased less and the amplitude of [Ca2+]i transient was much enhanced. This increase of [Ca2+]i was completely abolished by verapamil. DeltaG(exch) only recovered to a little above equilibrium (+1 kJ/mol). Inhibition of the Na+/K+-ATPase with ouabain entirely prevented the decrease of [Na+]i and caused a much larger increase of [Ca2+]i, which remained elevated; deltaG(exch) recovered to equilibrium and never returned to positive values. The rate of change of total cytosolic calcium was related to deltaG(exch), despite the fact that the calcium flux associated with the exchanger itself contributed only about 10%; SR related flux contributed by about 90% to the rate of change of total cytosolic calcium. In summary, reduction of [Na+]o causes reversal of the Na+/Ca2+-exchanger and its driving force deltaG(exch), a transient increase of [Ca2+]i and a decrease of [Na+]i. The influx of calcium associated with reversed deltaG(exch) triggers the release of calcium from SR. Both the decrease of [Na+]i and the increase of [Ca2+]i contribute to the recovery of deltaG(exch) to equilibrium. The time at which deltaG(exch) reaches equilibrium always coincides with the time to peak of [Ca2+]i transient. Activation of the Na+/K+-ATPase is required to reduce [Na+]i and recover deltaG(exch) to positive values in order to reduce [Ca2+]i. We conclude that deltaG(exch) is a major regulator of cytosolic calcium by interaction with SR.     

The effect of dietary vitamin D3 on the intracellular calcium gradient in mammalian colonic crypts

A physiological gradient in intracellular calcium ([Ca2+]i) has been hypothesized to exist along the colonic crypt base-mouth axis, which may be involved in the regulation of colonocyte proliferation, differentiation and apoptosis. In addition [Ca2+]i may be modulated by dietary vitamin D3 which is thought to be protective against colorectal cancer. CF1 mice were maintained for 6 weeks on a defined diet containing either high or low vitamin D3. A colonic crypt base-mouth [Ca2+]i gradient of 201 +/- 79 nM (mean +/- SEM, P < 0.05) was observed in animals maintained on a high vitamin D3 diet and was abolished in mice maintained on a low vitamin D3 diet. The [Ca2+]i gradient was independent of extracellular calcium and elevated levels of [Ca2+]i observed in the basal regions of the crypt in animals maintained on low levels of vitamin D3 were also associated with an increase in intracellular calcium stores. Therefore, a [Ca2+]i gradient exists in colonic crypts and is dependent on dietary vitamin D3.     

Lysophosphatidylcholine induces Ca2+-independent cellular injury attenuated by d-propranolol in rat cardiomyocytes

In isolated rat cardiomyocytes, exogenous lysophosphatidylcholine (LPC) (15 microM) increased the intracellular Ca2+ concentration (Ca2+]i) from 72 +/- 5 to 3042 +/- 431 nM accompanied by cell injury as indicated by the hypercontracture of the cells and the increase in creatine phosphokinase (CPK) release. In order to understand whether the cell injury induced by LPC was a consequence of the elevation of [Ca2+]i, the effect of LPC was examined in the Ca2+-free solution containing EGTA. Under the Ca2+ -free conditions, LPC did not increase [Ca2+]i, whereas it still inflicted injury on the cells in terms of cell-shape change and CPK release to the same degree as that under the Ca2+-present condition. Addition of ryanodine (10 microM) failed to prevent the changes in cell-shape and CPK release induced by LPC under both Ca2+-free and Ca2+-present conditions. Preincubation of the myocytes with d-propranolol (50 microM) inhibited the LPC-induced changes in cell-shape and CPK release under both Ca2+ -free and Ca2+ -present conditions (p < 0.05). Our study provides clear evidence that the cellular injury induced by LPC could be independent of the increase in [Ca2+]i, and the Ca2+-independent cellular injury induced by LPC could be attenuated by d-propranolol, although the mechanism remains unknown.     

Ca2+ signalling by endothelin receptors in rat and human cultured airway smooth muscle cells

1. The aim of the current study was to characterize the ET receptor subtypes in cultured airway smooth muscle cells derived from rat trachea and human bronchus using radioligand binding techniques and to investigate the coupling of ET receptors to intracellular calcium signalling mechanisms using endothelin receptor-selective agonists (sarafotoxin S6c) and antagonists (BQ-123, BQ-788) and digital image fluorescence microscopy. 2. Confluent rat airway smooth muscle cells in culture possessed a mixed ET receptor population (30% ETA : 70% ETB), with a density of approximately 3400+/-280 ETA and 8000+/-610 ETB receptors/cell (n = 3 experiments). The density of ETB, but not ETA receptors increased substantially in serum-containing medium. However, a 2-day period of serum deprivation, which inhibited cellular growth, substantially reduced ETB receptor density such that the ET receptor subtype proportions were approximately equal (55% ETA; 45% ETB) and similar to those previously observed in intact rat tracheal smooth muscle. 3. Challenge of rat airway smooth muscle cells in culture with endothelin- 1 elicited a concentration-dependent biphasic increase in [Ca2+]i (EC50: 16 nM), that comprised an initial transient peak [Ca2+]i increase (typically 350 nM) followed by a modest sustained component. The endothelin-1-induced biphasic [Ca2+]i increase was primarily due to ETA receptor activation, although a modest and inconsistent ETB response was observed. The ETA-mediated [Ca2+]i increase was due primarily to the mobilization of IP3-sensitive and to a lesser extent ryanodine-sensitive intracellular calcium stores. In contrast, ETB receptor activation was exclusively coupled to extracellular calcium influx. 4. Somewhat surprisingly, human airway smooth muscle cells in culture contained a homogeneous population of ETA receptors at a density of 6100+/-800 receptors cell(-1) (n = 3 experiments). Serum deprivation was without effect on either ET receptor subtype proportion or ETA receptor density. Challenge of human airway smooth muscle cells with endothelin-1 provoked a concentration-dependent increase in [Ca2+]i (EC50: 15 nM), with a peak [Ca2+]i increase to greater than 700 nM. Furthermore, the ETA-mediated calcium response in these human airway smooth muscle cells in culture was entirely dependent upon the mobilization of calcium from intracellular stores. 5. In summary, rat cultured tracheal airway smooth muscle cells contained both ETA and ETB receptors. ETA receptors, the numbers of which remained constant during cell growth, were linked to the release of Ca2+ from intracellular stores and a strong rise in [Ca2+]i in the majority of airway smooth muscle cells. In stark contrast, the numbers of ETB receptors increased significantly during cell growth, an effect that was diminished substantially by incubation in serum-free medium. Moreover, despite the greater number of ETB receptors, their activation in a small number of airway smooth muscle cells produced only a weak rise in [Ca2+]i, which appeared to be attributable to the influx of extracellular Ca2+. In contrast, the populations of ET receptors and their linkage to [Ca2+]i were markedly different in the human cultured airway smooth muscle cells used in the current study compared to that previously observed in intact human isolated bronchial smooth muscle.     

Atrial natriuretic peptide modulates the effect of angiotensin II on the concentration of free calcium in the cytosol of Mandin-Darby canine kidney cells

The effect of angiotensin II (ANG II) and atrial natriuretic peptide (ANP) on intracellular free calcium concentration [Ca2+]i was investigated in Mandin-Darby canine kidney (MDCK) cells in culture. Changes in [Ca2+]i were monitored fluorometrically with the Ca(2+)-sensitive probe fura-2/AM at 37 degrees C using a Perkin-Elmer LS-5 spectrofluorimeter (excitation 340/380 nm, slit 3 nm; emission 520 nm, slit 10 nm). MDCK cells exhibited a mean baseline [Ca2+]i of 98 +/- 10 nM. The addition of increasing concentrations of ANG II (1 pM to 1 microM) to the cell suspension led to a progressive increase in [Ca2+]i to 2-3 times basal levels. In contrast, addition of 1 microM ANP to the cell suspension led to a very rapid 60% decrease in [Ca2+]i. The addition of 1 pM to 1 microM ANG II immediately after 1 microM ANP caused an increase in [Ca2+]i which never exceeded the basal level in the absence of ANP. The data indicate that ANG II increases cell [Ca2+]i, as expected, and provide the new observation that ANP reduces [Ca2+]i in these cells. Furthermore, ANP reduces the increase in [Ca2+]i elicited by ANG II, thus modulating the effect of ANG II on [Ca2+]i.     

Synergistic effect and possible mechanisms of tumor necrosis factor and cisplatin cytotoxicity under moderate hyperthermia against gastric cancer cells

Photosensitization induces intracellular free calcium changes ([Ca2+]i) in some eukaryotic cell systems which either contribute to or protect against cell inactivation. We have investigated whether or not similar changes can be induced in prokaryotes. The skin bacterium Propionibacterium acnes was sensitized using protoporphyrin IX (PP IX) or 5-aminolevulinic acid (ALA). Exogenous ALA resulted in either a preferential accumulation of protoporphyrin (ALA-PP) or of coproporphyrin and/or uroporphyrin (ALA-CP/UP) in P. acnes. For PP IX or ALA-PP sensitization, exposure to broad-band red light resulted in an increase in [Ca2+]i. For ALA-PP sensitization, this increase was transient and [Ca2+]i returned to basal levels within 5-10 min after irradiation. However, the elevated [Ca2+]i levels obtained after PP IX sensitization were maintained for at least 1 h after irradiation. In both cases, the reduction in the external calcium concentration led to an enhancement in the cell survival, indicating that induced [Ca2+]i changes may participate in photoinactivation. Sensitization by hydrophilic coproporphyrin and/or uroporphyrin (ALA-CP/UP) did not affect the [Ca2+]i levels, but higher levels of cell inactivation were obtained. It therefore appears that damage to membrane-associated components is at least partly responsible for [Ca2+]i alterations after photosensitization.     

A novel, rapid and reversible method to measure Ca buffering and time-course of total sarcoplasmic reticulum Ca content in cardiac ventricular myocytes

This paper outlines a simple method of estimating both the Ca-buffering properties of the cytoplasm and the time-course of changes of sarcoplasmic reticulum (s.r.) Ca concentration during systole. The experiments were performed on voltage-clamped ferret single ventricular myocytes loaded with the free acid of fluo-3 through a patch pipette. The application of caffeine (10 mM) resulted in a Na-Ca exchange current and a transient increase of the free intracellular Ca concentration ([Ca2+]i). The time-course of change of total Ca in the cell was obtained by integrating the current and this was compared with the measurements of [Ca2+]i to obtain a buffering curve. This could be fit with a maximum capacity for the intrinsic buffers of 114+/-18 micromol l-1 and Kd of 0.59+/-0.17 microM (n=8). During the systolic rise of [Ca2+]i, the measured changes of [Ca2+]i and the buffering curve were used to calculate the magnitude and time-course of the change of total cytoplasmic Ca and thence of both s.r. Ca content and Ca release flux. This method provides a simple and reversible mechanism to measure Ca buffering and the time-course of both total cytoplasmic and s.r. Ca.     

Ventricular fibrillation-induced intracellular Ca2+ overload causes failed electrical defibrillation and post-shock reinitiation of fibrillation

Despite high efficacy, electrical defibrillation shocks can fail or ventricular fibrillation (VF) is reinitiated after the application of the initial shock. The goal of this study was to determine whether [Ca2+]i overload, induced by VF itself, can cause failed electrical defibrillation and post-shock reinitiation of VF. For this purpose, we simultaneously measured [Ca2+]i transients (assessed by indo-1 fluorescence) and defibrillation energies (assessed by a modified implantable cardioverter defibrillator) in intact perfused rat hearts during pacing-induced sustained VF (10 min) in the absence of ischemia. We found that increasing [Ca2+]i during VF (by increasing [Ca2+]o from 3 to 6 mM) increased the defibrillation threshold (DFT) from 1.9 +/- 0.6 to 3.5 +/- 0.5 J/g (P<0.05) and also increased the total defibrillation energy (TDE) required for stabilization of sinus rhythm from 15.6 +/- 7.7 to 48.6 +/- 7.42 J/g (P<0.05). In addition, both DFT and TDE correlated linearly with [Ca2+]i (r=0.69 and 0.83, P<0.05). Furthermore, shortening the duration of VF from 10 to 1.5 min tended to limit [Ca2+]i overload and decreased TDE. Finally, all successful defibrillation shocks led to a sudden reduction of VF-induced [Ca2+]i overload (-115 +/- 3%). In contrast, failed shocks did not alter [Ca2+]i. Incomplete reduction of [Ca2+]i overload after initially successful shocks were often followed by synchronized spontaneous [Ca2+]i oscillations and subsequent reinitiation of VF. In conclusion, the present study showed for the first time that VF-induced [Ca2+]i overload can cause failed electrical defibrillation and post-shock reinitiation of VF. Because VF inevitably causes [Ca2+]i overload, this finding might be a crucial mechanism of failed defibrillation and spontaneous reinitiation of VF.     

Calcium content of the sarcoplasmic reticulum in isolated ventricular myocytes from patients with terminal heart failure

Systolic [Ca2+]i-transients have been shown to be depressed in isolated ventricular myocytes from patients with terminal heart failure compared to controls. Experiments were performed in human ventricular cells to investigate whether this reduced systolic [Ca2+]i-transient may be due to a decreased Ca(2+)-content of the sarcoplasmic reticulum (SR). Single myocytes were isolated from left ventricular myocardium of patients with terminal heart failure undergoing cardiac transplantation. These results were compared to those obtained from cells of healthy donor hearts that were not suitable for transplantation for technical reasons. [Ca2+]i-transients were recorded from isolated cells under voltage clamp perfused internally with the Ca(2+)-indicator fura-2. The Ca(2+)-content of the SR was estimated by rapid extracellular application of caffeine (10 mM) to open the Ca(2+)-release channel of the SR and comparison of the caffeine-induced [Ca2+]i-transients in cells from patients with heart failure and from controls without heart failure. Upon steady-state depolarizations to +10 mV (maximum of the Ca(2+)-current), [Ca2+]i-transients in cells from patients with heart failure were significantly smaller than in myocytes from undiseased hearts (333 +/- 26 v 596 +/- 80 nM, P < 0.05). Application of caffeine caused a [Ca2+]i-transient that was always larger than during depolarization. Caffeine-induced [Ca2+]i-transients were significantly smaller in cells from diseased hearts compared with controls (970 +/- 129 v 2586 +/- 288 nM, P < 0.01). A positive correlation was found between left ventricular ejection fraction and caffeine-induced [Ca2+]i-transients in these cells. It is concluded, that depressed [Ca2+]i-transients in myocytes from patients with heart failure may be caused by a decreased Ca(2+)-content of the SR possibly due to an altered Ca(2+)-ATPase activity in these hearts. It is not necessary to postulate an additional defect of the Ca(2+)-release function of the SR to account for the alterations of intracellular (Ca2+]i-handling.     

Ca2+ transients in cardiac myocytes measured with high and low affinity Ca2+ indicators

Intracellular calcium ion ([Ca2+]i) transients were measured in voltage-clamped rat cardiac myocytes with fura-2 or furaptra to quantitate rapid changes in [Ca2+]i. Patch electrode solutions contained the K+ salt of fura-2 (50 microM) or furaptra (300 microM). With identical experimental conditions, peak amplitude of stimulated [Ca2+]i transients in furaptra-loaded myocytes was 4- to 6-fold greater than that in fura-2-loaded cells. To determine the reason for this discrepancy, intracellular fura-2 Ca2+ buffering, kinetics of Ca2+ binding, and optical properties were examined. Decreasing cellular fura-2 concentration by lowering electrode fura-2 concentration 5-fold, decreased the difference between the amplitudes of [Ca2+]i transients in fura-2 and furaptra-loaded myocytes by twofold. Thus, fura-2 buffers [Ca2+]i under these conditions; however, Ca2+ buffering is not the only factor that explains the different amplitudes of the [Ca2+]i transients measured with these indicators. From the temporal comparison of the [Ca2+]i transients measured with fura-2 and furaptra, the apparent reverse rate constant for Ca2+ binding of fura-2 was at least 65s-1, much faster than previously reported in skeletal muscle fibers. These binding kinetics do not explain the difference in the size of the [Ca2+]i transients reported by fura-2 and furaptra. Parameters for fura-2 calibration, Rmin, Rmax, and beta, were obtained in salt solutions (in vitro) and in myocytes exposed to the Ca2+ ionophore, 4-Br A23187, in EGTA-buffered solutions (in situ). Calibration of fura-2 fluorescence signals with these in situ parameters yielded [Ca2+]i transients whose peak amplitude was 50-100% larger than those calculated with in vitro parameters. Thus, in vitro calibration of fura-2 fluorescence significantly underestimates the amplitude of the [Ca2+]i transient. These data suggest that the difference in amplitude of [Ca2+]i transients in fura-2 and furaptra-loaded myocytes is due, in part, to Ca2+ buffering by fura-2 and use of in vitro calibration parameters.     

Histamine-induced calcium released from cultured human mucosal microvascular endothelial cells from nasal inferior turbinate

Changes in cytosolic Ca2+ concentration ([Ca2+]i) in cultured human mucosal microvascular endothelial cells (HMMECs) from nasal inferior turbinate were measured using a fluorescent Ca(2+)-sensitive dye, fura-2, and photometric fluorescence microscopy. Histamine caused a transient increase in intracellular free Ca2+ in cell populations and in individual cells, followed by a decrease to a sustained elevation. Histamine (100 microM) elevated [Ca2+]i in HMMECs up to 563 +/- 20 nM from a resting level of 60 +/- 45 nM (means +/- SD, n = 31). Promethazine (a histamine H1 receptor antagonist) inhibited [Ca2+]i increase during histamine stimulation, whereas cimetidine (a H2 receptor antagonist) and thioperamide (a H3 receptor antagonist) showed no inhibition. These results suggest that the histamine increase [Ca2+]i in HMMECs induces both a Ca2+ release from stores and a Ca2+ influx through activation of the H1 receptor.     

Effect of alkalinization of cytosolic pH by amines on intracellular Ca2+ activity in HT29 cells

The effect of secondary, tertiary and quaternary methyl- and ethylamines on intracellular pH (pHi) and intracellular Ca2+ activity ([Ca2+]i) of HT29 cells was investigated microspectrofluorimetrically using pH- and Ca2+- sensitive fluorescent indicators, [i.e. 2', 7'-biscarboxyethyl-5(6)-carboxyfluorescein (BCECF) and fura-2 respectively]. Membrane voltage (Vm) was studied by the patch-clamp technique. Secondary and tertiary amines led to a rapid and stable concentration-dependent alkalinization which was independent of their pKa value. Trimethylamine (20 mmol/l) increased pHi by 0.78 +/- 0.03 pH units (n = 9) and pH remained stable for the application time. Removal led to an undershoot of pHi and a slow and incomplete recovery: pHi stayed 0.26 +/- 0.06 pH units more acid than the resting value. The quaternary amines, tetramethyl- and tetraethylamine were without influence on pHi. All tested secondary and tertiary amines (dimethyl-, diethyl-, trimethyl-, and triethyl-amine) induced a [Ca2+]i transient which reached a peak value within 10-25 s and then slowly declined to a [Ca2+]i plateau. The initial Delta[Ca2+]i induced by trimethylamine (20 mmol/l) was 160 +/- 15 nmol/l (n = 17). The [Ca2+]i peak was independent of the Ca2+ activity in the bath solution, but the [Ca2+]i plateau was significantly lower under Ca2+-free conditions and could be immediately interrupted by application of CO2 (10%; n = 6), a manoeuvre to acidify pHi in HT29 cells. Emptying of the carbachol- or neurotensin-sensitive intracellular Ca2+ stores completely abolished this [Ca2+]i transient. Tetramethylamine led to higher [Ca2+]i changes than the other amines tested and only this transient could be completely blocked by atropine (10(-6) mol/l). Trimethylamine (20 mmol/l) hyperpolarized Vm by 22.5 +/- 3.7 mV (n = 16) and increased the whole-cell conductance by 2.3 +/- 0.5 nS (n = 16). We conclude that secondary and tertiary amines induce stable alkaline pHi changes, release Ca2+ from intracellular, inositol-1,4, 5-trisphosphate-sensitive Ca2+ stores and increase Ca2+ influx into HT29 cells. The latter may be related to both the store depletion and the hyperpolarization.     

Flow cytometric study of mitochondrial dysfunction after AMPA receptor activation

The effect of AMPA-receptor stimulation on MMP and on the concentration of intracellular calcium ([Ca2+]i) was studied in dissociated CGC from rat pups, by flow cytometry. In the presence of cyclothiazide, AMPA induced a sodium-independent decrease in MMP up to 30.7+/-2.5%. This effect was antagonized by CNQX and NBQX. Mepacrine and dibucaine reversed the effect of AMPA on MMP, suggesting that it is mediated by a release of arachidonic acid. AMPA alone induced a slight (about 7%) increase in [Ca2+]i. In the presence of cyclothiazide, AMPA induced a concentration-dependent [Ca2+]i increase up to 29.10+/-2.10% that was not reversed by flunarizine. This increase was similar to that observed in a Na+-free medium, and was antagonized by CNQX and NBQX, but not by MK-801. Mitochondria play a key role in the modulation of [Ca2+]i since a significant [Ca2+]i increase was found in the presence of FCCP. On the other hand, the dantrolene-sensitive calcium pools do not participate in the [Ca2+]i increase induced by stimulation of AMPA receptors. It is concluded that when AMPA-receptor desensitization is blocked, a decrease in MMP and an increase in [Ca2+]i occurs, which could be additional events to potentiate neuronal cell death induced by glutamate.     

P2 receptor-mediated signal transduction in Ehrlich ascites tumor cells

The mechanisms, by which the P2 receptor agonists adenosine 5'-triphosphate (ATP) and uridine 5'-triphosphate (UTP) evoke an increase in the free cytosolic calcium concentration ([Ca2+]i) and in intracellular pH (pHi), have been investigated in Ehrlich ascites tumor cells. The increase in [Ca2+]i evoked by ATP or UTP is abolished after depletion of intracellular Ca2+ stores with thapsigargin in Ca2+-free medium, and is inhibited by U73122, an inhibitor of phospholipase C (PLC), indicating that the increase in [Ca2+]i is primarily due to release from intracellular, Ins(1,4,5)P3-sensitive Ca2+ stores. ATP also activates a capacitative Ca2+-entry pathway. ATP as well as UTP evokes a biphasic change in pHi, consisting of an initial acidification followed by alkalinization. Suramin and 4,4'-diisothiocyano-2,2'-stilbene-disulfonic acid (DIDS) inhibit the biphasic change in pHi, apparently by acting as antagonists at P2 receptors. The alkalinization evoked by the P2 receptor agonists is found to be due to activation of a 5'-(N-ethyl-N-isopropyl)amiloride (EIPA)-sensitive Na+/H+ exchanger. ATP and UTP elicit rapid cell shrinkage, presumably due to activation of Ca2+ sensitive K+ and Cl- efflux pathways. Preventing cell shrinkage, either by incubating the cells at high extracellular K+ concentration, or by adding the K+-channel blocker, charybdotoxin, does not affect the increase in [Ca2+]i, but abolishes the activation of the Na+/H+ exchanger, indicating that activation of the Na+/H+ exchanger is secondary to the Ca2+-induced cell shrinkage.     

Evidence for an increased intracellular free calcium concentration in platelets of bronchial asthma patients

The pathogenesis of bronchial asthma is not yet fully understood. Recently much attention has been given to the hypothesis that intracellular free calcium ([Ca2+]i) metabolism is abnormal in various diseases. In this study we investigated whether [Ca2+]i exists abnormally in subjects with bronchial asthma. The [Ca2+]i in 32 treated or untreated subjects with bronchial asthma were compared with 63 normal subjects. Resting levels of [Ca2+]i were estimated by loading the fluorescent indicator Fura-2 in washed platelets. The [Ca2+]i level in the control subjects was 129.7 +/- 18.0 nM (mean +/- SD). However, in that of the bronchial asthma patients was 152.7 +/- 44.1 nM, significantly higher than that of the control subjects (p < 0.05). It is well recognized that an increase of [Ca2+]i in vascular smooth muscle involves contraction. The findings suggest that the same phenomenon is quite possible in the tracheal smooth muscle and that it plays an important role in the pathogenesis of bronchial asthma.